RNA interference (RNAi) was originally described as a gene silencing mechanism triggered by the experimental introduction of double stranded (ds)RNA into the nematode C. elegans (Fire et al., 1998). The term RNAi is now used to refer to a diverse set of gene-regulatory mechanisms that share common features, including the involvement of a short 21-30 nucleotide (nt) long RNA and a protein cofactor of the Argonaute (RNase H-related) protein family. As an experimental tool, RNAi is of broad relevance to basic medical research in numerous fields, and RNAi therapeutics are now under development for several clinical applications. Furthermore, RNAi-related mechanisms function in conserved gene-regulatory pathways that are of basic and fundamental importance to human cellular and developmental biology. Remarkably, RNAi-related mechanisms in C. elegans keep inventory of all mRNAs and license gene expression in the germline, passing this information via the egg and sperm from one generation to the next. Distinct Argonaute pathways function in the transgenerational inheritance of small-RNA signals that constitute the CSR-1 self/protective (RNAa pathway) and the WAGO non-self/silencing (RNAe pathway). A third Argonaute pathway, the PRG-1/Piwi pathway scans germline mRNA for foreign sequences. In Aim 1, we explore how these Argonaute pathways identify their targets and recruit downstream factors. In Aim 2, we investigate how these Argonaute pathways interact to mediate genome-wide transcriptional surveillance. And finally, in Aim 3, we describe genetic screens that will allow us to identify new genes required for RNAi and the related RNAe and RNAa pathways. The ability to combine classical genetics with technology, including deep-sequencing, CRISPR-mediated genome editing, and proteomics, make C. elegans an ideal system for these studies. The mechanisms and protein families that mediate RNAi are highly conserved in animals and therefore insights from the proposed studies will be directly relevant to human biology and disease. These studies will shed light on an ancient gene-regulatory mechanism whose correlates in humans are likely to play important conserved roles in the protection of the human genome and the maintenance of stem cells.